We propose to continue our work identifying and validating biomarkers - particularly hormonal markers - that predict risk of invasive breast cancer in postmenopausal women. Using a prospective nested case-control design, we plan to analyze blood samples collected from the 32,826 participants in the Nurses' Health Study (NHS) who provided a blood sample in 1989-90 and, for 18,743 of these women, a second sample in 1999- 2000. We propose to evaluate markers from several inter-related pathways to determine their role in cancer risk; a number of these aims are entirely new, while others extend our work in the most promising areas from the current grant cycle. Specifically, we will evaluate the role of several matrix metalloproteins (MMPs), estrogen metabolites, prolactin as assessed by a novel bioassay, 25(OH) vitamin D, estradiol and testosterone in relation to risk; for prolactin and vitamin D, we will assess whether the plasma level / breast cancer relation varies by protein expression in tumor tissue (e.g., prolactin receptor expression). We also will assess the importance of timing of exposure in carcinogenesis using two blood samples collected ten years apart. Further, we will provide the first detailed assessment of whether plasma hormones, mammographic density and several well-confirmed gene polymorphisms contribute importantly to risk prediction models for postmenopausal estrogen receptor positive (ER+) breast cancer. Such models may be useful in identifying women at high risk of breast cancer who would most benefit from chemoprevention or increased screening - yet no prior study has evaluated this range of risk factors and few have assessed ER+ tumors specifically (although currently approved chemopreventives only decrease risk of ER+ breast cancer). The ongoing NHS Program Project grant will provide follow-up and documentation of breast cancer outcomes in addition to providing information on important covariates, substantially increasing the cost-effectiveness of the proposed project.